Immunoaffinity chromatography is a valuable protein separation technique that takes advantage of the binding specificity between an antibody and its antigen. (For a review see reference [1].) This purification method, however, is inherently limited due to the tight binding between the antibody and its antigen. In a great many instances, a high concentration of denaturing reagents, or extreme pH values, or both, is required to dissociate the antibody-antigen complex. As a result, the process of dissociating the antibody from its antigen often yields a denatured protein product. Where the antigen to be isolated is a multi-subunit protein complex, these concerns are more troublesome because the conditions must be sufficiently harsh to dissociate the antibody-antigen complex, but not so harsh as to dissociate the protein complex itself.
The term “immunoaffinity chromatography” as used herein designates any method that uses immobilized antibodies in affinity chromatography. Immunoaffinity chromatography is one of the most powerful protein purification procedures currently available. When successfully executed, the exquisite specificity and high affinity of the antibody-antigen interaction lead to the highly selective absorption of target proteins to the immobilized antibodies. Thus, by applying a protein mixture to a suitable antibody immobilized on a resin, washing off unbound or weakly bound material, and then eluting the antigenic protein with appropriate elution agents, both purification (often of greater than 1,000-fold) and simultaneous concentration can be achieved. As a result, immunoaffinity chromatography has proven extremely useful for both biochemical laboratory-scale and large-scale protein purification [18, 19, 20].
Two types of antibodies are used in immunoaffinity chromatography, polyclonal antibodies and monoclonal antibodies. On one hand, polyclonal antibodies, obtained by immunizing a rabbit or goat and purifying the immunoglobulin fraction from the resulting serum, are mixtures of antibodies with a variety of specificities and binding properties. Polyclonal antibodies are capable of binding to various parts (epitopes) of the protein used as the immunogen. Polyclonal antibodies are relatively easy to produce but suffer from several disadvantages when used in immunoaffinity chromatography. Most notably, polyclonal antibodies are heterogeneous with respect to epitope specificity and binding properties. Therefore, great care must be taken to immunize the animal with highly pure protein to avoid raising unwanted antibodies to minor impurities in the inoculation preparation. Also, the antibody preparation is not completely reproducible from one immunized animal to another. (Each animal has a similar but not identical immune response to the inoculant.) This makes it impossible to obtain large quantities of antibodies with consistent properties.
On the other hand, monoclonal antibodies, while more difficult and expensive to produce than polyclonal antibodies, have several advantages for use in immunoaffinity chromatography. Monoclonal antibodies can be produced with smaller quantities of less purified immunogen. Once a hybridoma line is established, it can be used to produce a potentially unlimited supply of antibody with reproducible properties. Most importantly, the antibody binds to a single epitope on the antigen and thus has homogeneous binding and dissociation (i.e., elution) properties.
As noted earlier, because of the strength of the antigen-antibody interaction, it is usually difficult to elute the antigen from an immunoaffinity column. It is not uncommon to employ quite harsh elution conditions, such as extreme pH values (pH 3 or 10), denaturing agents (8 M urea or 6 M guanidinium hydrochloride), or chaotropic salts (3 M KSCN) that disrupt protein structure. The elution conditions often damage labile proteins, especially multi-subunit enzymes, resulting in very low yields of active, purified protein. The harsh conditions also decrease the lifetime of the antibody column.
Monoclonal antibodies with a variety of special properties have been isolated in an effort to avoid the requirements of harsh elution conditions. In a very early study, monoclonal antibodies were screened for those that required less extreme pH's for elution of the antigen; e.g., elution at pH 4.5 instead of pH 3 [21]. Monoclonal antibodies were also found that bind to antigen in the presence of Ca+2 and can be eluted with the calcium chelator EGTA [22].
A specific sub-type of monoclonal antibody, called a “polyol-responsive” monoclonal antibody (hereinafter “PR-mAb”) has properties that are ideally suited for use in immunoaffinity chromatography. A PR-mAb binds very tightly to its antigen under many standard conditions, but releases the antigen when eluted under very mild, non-denaturing conditions, namely an aqueous buffer at neutral pH supplemented with a low molecular-weight polyhydroxylated compound (i.e., a polyol), such as ethylene glycol or propylene glycol, and a nonchaotropic salt such as NaCl or ammonium sulfate. Generally salt alone or polyol alone do not cause antigen elution, although some mAbs respond to polyol alone. The resulting purified proteins are active and multi-subunit complexes are retained. (For reviews see [2, 3, and 4].) PR-mAb immunoaffinity chromatography has been used successfully to purify large multi-protein complexes such as RNA polymerase (RNAP) [5, 6, 7].
Six different PR-mAbs have been employed in immunoaffinity chromatography in the lab of the present inventors [3, 4]. The PR-mAb identification procedure set forth in reference [4] has also been used in other labs to isolate PR-mAbs for gently purifying a variety of proteins and protein complexes [8, 9, 10]. However, PR-mAbs make up only about 5% to perhaps 10% of the antibody repertoire in the mouse [7]. Moreover, there are far more proteins to be isolated than there are corresponding PR-mAbs. Thus, there remains a long-felt and unmet need to expand the range of protein targets that can be purified by immunoaffinity chromatography using PR-mAbs.